CTLA4 +49AG (rs231775) and CT60 (rs3087243) gene variants are not associated with alopecia areata in a Mexican population from Monterrey Mexico

Abstract Background: Alopecia areata is an autoimmune disease that produces non-scarring hair loss around the body. Gene variants of the cytotoxic T-lymphocyte antigen 4 (CTLA4) gene, a negative regulator of T-cell response, have been associated with a predisposition to autoimmune diseases in different populations; however, the involvement of these genetic variants in the development of AA is controversial. Objective: The present study evaluated the potential association of two CTLA4 gene variants with alopecia areata in a Mexican population. Methods: We genotyped +49AG (rs231775) and CT60 (rs3087243) variants in 50 AA patients and 100 healthy control participants through PCR-RFLP. Results: No statistical difference was observed for either of the gene variants regarding allele or genotype frequencies between AA patients and the controls when the parameters of family/personal history of autoimmune diseases or gender were considered (p > 0.05). Study limitations: Small sample size of patients and the data were obtained from Northeast Mexico population. Conclusion: The genetic variants rs231775 and rs3087243 of the CTLA4 gene are not a risk factor for the development of alopecia areata in the analyzed Mexican population.

Cytotoxic T Lymphocyte-associated Antigen 4 Polymorphisms Correlated with Graves' Disease in Patients of Han Ethnicity in Yunnan, China / Polimorfismos del antígeno 4 del linfocito T citotóxico correlacionados con la enfermedad de Graves en pacientes de la etnia Han en Yunnan, China

ABSTRACT Objective: To investigate the correlations between polymorphisms at position 49 in exon 1 and position 318 in the promoter of the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) gene and autoimmune thyroid diseases in a Han Chinese population. Methods: Polymerase chain reaction-restriction fragment length polymorphism was utilized. The MseI and BbvI restriction endonucleases were used to detect and analyse position 49 in exon 1 and position 318 in the promoter as well as the T/C alleles of the CTLA-4 gene in peripheral blood samples from 112 patients with Graves' disease (GD), 101 with Hashimoto's thyroiditis (HT) and 100 healthy individuals. Results: At position 49 of exon 1, the frequencies of the GG genotype and the G allele in the GD group (χ2 = 12.147; p = 0.002) were statistically significantly higher than those in the control group (χ2 = 9.925; p = 0.002), while no statistically significant differences were found between the frequencies of the GG genotype and the G allele in the HT group (χ2 = 1.195; p = 0.550) and those in the control group (χ2 = 0.984; p = 0.321). No statistically significant differences in the promoter (−318) or the T/C alleles were observed among the three groups. Position 49 in the 17th codon of exon 1 of the CTLA-4 gene may be a candidate susceptibility marker in patients of Han ethnicity with GD. Conclusion: This finding helps us to better understand the genetic risks for GD and provides a direction for targeted gene therapy.

RESUMEN Objetivo: Investigar las correlaciones entre los polimorfismos en la posición 49 en el exón 1 y la posición 318 en el promotor del gen del antígeno 4 asociado al linfocito T citotóxico (CTLA-4), con las enfermedades autoinmunes de la tiroides en una población China de Han. Métodos: Se utilizó la reacción en cadena de la polimerasa-polimorfismo de la longitud de los fragmentos de restricción. Las endonucleasas de restricción de MseI y BbvI se utilizaron para detectar y analizar la posición 49 en el exón 1 y la posición 318 en el promotor, así como los alelos T/C del gen CTLA-4 en muestras de sangre periférica de 112 pacientes con enfermedad de Graves (EG), 101 con tiroiditis de Hashimoto (TH) y 100 individuos sanos. Resultados: En la posición 49 de exón 1, las frecuencias del genotipo GG y el alelo G en el grupo de EG (χ2 = 12.147; p = 0.002) fueron estadísticamente significativamente más altas que las del grupo de control (χ2 = 9.925; p = 0.002), pero no se encontraron diferencias estadísticamente significativas entre las frecuencias del genotipo GG y el alelo G en el grupo de TH (χ2 = 1.195; p = 0.550) y las del grupo de control (χ2 = 0.984; p = 0.321). No se observaron diferencias estadísticamente significativas en el promotor (−318) ni en los alelos T/C entre los tres grupos. La posición 49 en el codón17.° del exón 1 del gen CTLA-4 puede ser un marcador de susceptibilidad candidato en pacientes de la etnia Han con EG. Conclusión: Este hallazgo nos ayuda a comprender mejor los riesgos genéticos de la EG y ofrece una dirección para la terapia génica dirigida.

Polimorfismos de un solo nucleótido representativos para los alelos clásicos del antígeno leucocitario humano en familias antioqueñas con diabetes mellitus tipo 1 / Classical HLA alleles tag SNP in families from Antioquia with type 1 diabetes mellitus

Resumen Introducción. La región del antígeno leucocitario humano (Human Leukocyte Antigen, HLA) se ha asociado claramente con enfermedades autoinmunitarias, como la diabetes mellitus de tipo 1. Los polimorfismos representativos de un solo nucleótido (tag Single Nucleotide Polymorphism, tag SNP) constituyen una forma alternativa de evaluar los alelos clásicos del HLA. En la población europea se ha reportado un grupo de tag SNP para múltiples alelos clásicos relacionados con la predisposición o la resistencia frente a dicha enfermedad. Objetivo. Validar la metodología basada en los tag SNP enfocada en la inferencia de alelos HLA clásicos, y evaluar su asociación con la diabetes mellitus de tipo 1 en una muestra de familias antioqueñas. Materiales y métodos. Se estudió una muestra de 200 familias antioqueñas con uno a dos hijos afectados por diabetes mellitus de tipo 1. Se genotipificaron 13 SNP mediante el ARMS-PCR (Amplification Refractory Mutation System-Polymerase Chain Reaction) con cuatro iniciadores, o mediante la PCR-RFLP (PCR-Restriction Fragment Length Polymorphism). Además, se evaluó la validez de los tag SNP de 1.000 genomas reportados en europeos en una muestra de 60 individuos de la población colombiana de Medellín. Se hicieron las pruebas de desequilibrio de la transmisión, de desequilibrio de ligamiento y de equilibrio de Hardy-Weinberg. Resultados. En la población de estudio no se encontró suficiente desequilibrio de ligamiento entre los SNP y los alelos clásicos evaluados, por lo cual no fue posible inferir los alelos clásicos del HLA para el conjunto de familias con diabetes mellitus de tipo 1. El estudio de asociación evidenció que esta región aporta factores tanto de riesgo como de protección para el desarrollo de la enfermedad. Los tag SNP apropiados para la muestra de estudio se determinaron usando los SNP ubicados en la región HLA en la base de datos del 1000 Genomes Project en la mencionada población. Conclusiones. Los patrones de desequilibrio de ligamiento en la población estudiada fueron diferentes a los reportados para la población europea. A pesar de esto, se encontró evidencia clara sobre el papel de la región HLA en el riesgo de padecer diabetes mellitus de tipo 1 en la población de estudio.

abstract Introduction: The HLA region strongly associates with autoimmune diseases, such as type 1 diabetes. An alternative way to test classical HLA alleles is by using tag SNP. A set of tag SNP for several classical HLA alleles has been reported as associated with susceptibility or resistance to this disease in Europeans. Objective: We aimed at validating the methodology based on tag SNP focused on the inference of classical HLA alleles, and at evaluating their association with type 1 diabetes mellitus in a sample of 200 families from Antioquia. Materials and methods: We studied a sample of 200 families from Antioquia. Each family had one or two children with T1D. We genotyped 13 SNPs using tetra-primer ARMS-PCR or PCRRFLP. In addition, we tested the validity of the tag SNP reported for Europeans in 60 individuals from a population of Colombians living in Medellín (CLM) from the 1000 Genomes Project database. Statistical analyses included the Hardy-Weinberg equilibrium, the transmission disequilibrium and the linkage disequilibrium tests. Results: The linkage disequilibrium was low in reported tag SNP and classical HLA alleles in this CLM population. Association analyses revealed both risk and protection factors to develop type 1 diabetes mellitus. Appropriate tag SNPs for the CLM population were determined by using the genotype information available in the 1000 Genome Project database. Conclusions: Although linkage disequilibrium patterns in this CLM population were different from those reported in Europeans, we did find strong evidence of the role of HLA in the development of type 1 diabetes mellitus in the study population.

Construction of a fusion plasmid containing the PSCA gene and cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and its anti-tumor effect in an animal model of prostate cancer

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is a negative regulator of T cell activation, which competes with CD28 for B7.1/B7.2 binding, and which has a greater affinity. Fusion of specific antigens to extracellular domain of CTLA4 represents a promising approach to increase the immunogenicity of DNA vaccines. In this study, we evaluated this interesting approach for CTLA4 enhancement on prostate stem cell antigen (PSCA)-specific immune responses and its anti-tumor effects in a prostate cancer mouse model. Consequently, we constructed a DNA vaccine containing the PSCA and the CTLA-4 gene. Vaccination with the CTLA4-fused DNA not only induced a much higher level of anti-PSCA antibody, but also increased PSCA-specific T cell response in mice. To evaluate the anti-tumor efficacy of the plasmids, murine models with PSCA-expressing tumors were generated. After injection of the tumor-bearing mouse model, the plasmid carrying the CTLA4 and PSCA fusion gene showed stronger inhibition of tumor growth than the plasmid expressing PSCA alone. These observations emphasize the potential of the CTLA4-fused DNA vaccine, which could represent a promising approach for tumor immunotherapy.

Phenotypic Characteristics of PD-1 and CTLA-4 Expression in Symptomatic Acute Hepatitis A

BACKGROUND/AIMS: The immunoregulatory molecules programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) are associated with the dysfunction of antiviral effector T-cells, which leads to T-cell exhaustion and persistent viral infection in patients with chronic hepatitis C and chronic hepatitis B. Little is known about the role of PD-1 and CTLA-4 in patients with symptomatic acute hepatitis A (AHA). METHODS: Peripheral blood mononuclear cells were isolated from seven patients with AHA and from six patients with nonviral acute toxic hepatitis (ATH) during the symptomatic and convalescent phases of the respective diseases; five healthy subjects acted as controls. The expression of PD-1 and CTLA-4 on T-cells was measured by flow cytometry. RESULTS: PD-1 and CTLA-4 expression during the symptomatic phase was significantly higher in the T-cells of AHA patients than in those of ATH patients or healthy controls (PD-1: 18.3% vs 3.7% vs 1.6%, respectively, p<0.05; CTLA-4: 23.5% vs 6.1% vs 5.9%, respectively, p<0.05). The levels of both molecules decreased dramatically during the convalescent phase of AHA, whereas a similar pattern was not seen in ATH. CONCLUSIONS: Our findings are consistent with a viral-protective effect of PD-1 and CTLA-4 as inhibitory molecules that suppress cytotoxic T-cells and thereby prevent the destruction of virus-infected hepatocytes in AHA.

Analysis of cytotoxic T-lymphocyte-associated antigen-4 and MMP-9 genes’ methylation and their expression profiles with risk of non-alcoholic fatty liver disease.

OBJECTIVE: To investigate the effect of promoter methylation of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) gene and matrix metalloproteinases (MMPs) on the risk of non-alcoholic fatty liver disease (NAFLD). MATERIALS AND METHODS: CTLA-4 and MMP-9 promoter methylation were investigated using a methylation-specific polymerase chain reaction (MS-PCR) in blood samples taken from 80 NAFLD individuals and 95 healthy controls. The expression levels of CTLA-4 and MMP-9 were also assessed in 10 blood and 9 liver tissues mRNA samples from NAFLD patients. These cases were compared to the blood (n = 10) samples of healthy controls with real-time quantitative reverse transcriptase PCR. RESULTS: No significant relationship was found for methylation of CTLA-4 and MMP-9 between cases and controls. The relative expression of CTLA-4 and MMP-9 mRNA in NAFLD was not significantly different compared to healthy control samples. CONCLUSION: For the first time, our outcomes indicate that the methylation status of CTLA-4 and MMP-9 genes has no significant function on the process of NAFLD.